Figure 3. Minimal cross-modular promoter leakiness was observed with dual luciferase assay.

3A, Schematic illustration of the three-module construct. The abbreviations of depicted elements were annotated in Table 1. 3B, and 3C, response of firefly (3B) and renilla (3C) luciferase activities to increasing concentrations of doxycycline; the above construct was stably transfected into HCT116 cells and selected with 1 µg/ml puromycin. Surviving cells were seeded in 24-well plates the day prior to treatment to reach around 80% confluence at the time of treatment; vehicle or doxycycline of indicated concentrations were added for incubation for 24 hours at 37°C before dual luciferase assay. Each treatment was triplicated. Results were represented as relative light units normalized to protein content (RLU/µg). Comparisons were made between results in samples treated with indicated doxycycline concentration and those treated with vehicle only (doxycycline 0 µg/ml). *, p<0.05; **, p<0.01.